FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, SUPPLEMENT NO. 1, S134-S137
Correction by dietary linoleic acid of rat adipocyte metabolic disorders in essential fatty acid deficiency I. HARANT, C. CARPÉNɆ, J. GARCIA, J. P. THOUVENOT and J. GHISOLFI Groupe d'Etudes en Nutrition Infantile, CEDEM, Hôpital Purpan, 31059 Toulouse, France, and † INSERM U 317, Institut de Physiologie, rue F. Magendie, 31400, Toulouse, France
Introduction
Essential fatty acid (EFA) deficiency occurs in some infant nutritional or digestive diseases such as biliary atresia (Gourley et al. 1982). In order to determine the adequate level of linoleic acid (LA) intake necessary to prevent EFA deficiency symptoms, the effects of LA supplementation on fat cell lipid composition and lipolytic/lipogenic activities were studied in rats submitted to an EFA-deficient diet. Moreover, fatty acid (FA) composition of adipose tissue, a key organ for FA storage, was compared to the commonly used plasma index of EFA deficiency: eicosatrienoate/arachidonate ratio in erythrocyte phospholipids (PL). Materials and methods
Four-week-old rats were submitted to a diet containing 5% lipids (% dry weight) constituted of either peanut- and rape-oil (controls, LA = 2-5% of calorie intake) or the saturated FA: stearate and palmitate (treated, LA = 0-02% of calorie intake). After 9 weeks, the treated rats were divided into three groups and their diets supplemented with different amounts of LA: 1, 2-5 and 5% of calorie intake for 6 weeks. The following parameters were measured: Body weight, adipose mass and blood levels of various metabolites. FA composition of erythrocyte PL: the PL fractions were separated by thinlayer chromatography (Garcia et al. 1986). FA composition of stored triglycerides (TG) and membrane PL of isolated adipocytes: neutral lipids were separated from the PL by the method of Juaneda (1985). After transesterification, FA of erythrocytes and adipocytes were analysed by gas-chromatography (Garcia et al. 1986). In vitro metabolic activity of adipocytes: lipolytic activity (Rodbell 1964) and lipogenic activity (incorporation of (3-3H)-glucose into lipid metabolites according to Moody et al. 1974). Results and discussion
The unsaturated FA-deficient diet induced, as quickly as 2 weeks of treatment, a significant reduction in body weight gain of the rats. After 9 weeks of treatment, body weight was 334 ± 7 g for the treated compared with 421 ± 6 g for the controls (n = 30, p < 0-001). This diminution of growth is a symptom of EFA deficiency as 0265-203X/90 $3.00© 1990 Taylor & Francis Ltd.
Linoleic acid and rat adipocyte metabolic disorders
S135
already reported (Yamanaka et al. 1981, Kritchevsky etal. 1988). The adipose tissue weight was also reduced. After 5 weeks of treatment it was reduced by 50% and more severely after 9 weeks (9-3 ± 1-3 g versus 24-0 ± 2-2 g, p < 0-001). The mass of other organs (brain, liver, heart, pancreas, kidney) were not so affected by EFA deficiency (Berdanier and Baltzell 1986, Kritchevsky et al. 1988, Yamanaka et al. 1981). So, it appears that the EFA deficiency had a specific inhibitory effect on the adipose tissue development. FA composition of erythrocyte PL showed an important decrease of linoleic and arachidonic acid percentages in treated rats. Conversely, the eicosatrienoic acid percentage increased. So, the eicosatrienoate/arachidonate ratio was higher after nine weeks in treated than in control (1-5 ±0-2% versus 0-010 ± 0-002%, p < 0-001). These modifications of erythrocyte FA composition clearly indicated the onset of an EFA deficiency (Yamanaka et al. 1981). In adipocytes, FA composition of stored TG and membranes PL followed the same evolution pattern (C20:3w9/C20:4co6 = 1 -8 ± 0-3% versus 0-08 ± 0-01% for PL in treated and controls, p < 0-001, n = 4). In order to determine if the fat stores reduction was due to an increased lipolytic activity or to a decreased lipogenesis accompanying the modifications of adipocyte membrane composition, these activities were studied in isolated fat cells. No significant change was found in the responses to various lipolytic agents (isoproterenol, forskolin) but there was an increase in sensitivity to ACTH for deficient rats at 5 and 9 weeks of treatment (table 1). This increase of lipolysis, observed in vitro, is in good agreement with the depletion of adipose tissue observed in vivo. However, it is insufficient to explain the adipose mass reduction of treated rats. By contrast, basal and insulin-stimulated lipogenesis were significantly increased in deficient rats (table 2). This observation in vitro is apparently in contradiction with the in vivo fat store depletion observed in the treated group since fat cells with an increased lipogenic activity would correspond to an enlargement of adipose tissue. So, the endocrine or metabolic environment of adipose tissue might be involved in this slimming effect of EFA deficiency rather than a decline in metabolic rate of fat cells. On this point, it must be noted that deficient rats were
Table 1. Effect of unsaturated fatty acid deficiency on lipolytic response of rat adipocytes to synacthen. Percentage of maximal lipolysis Addition to medium synacthen /ig/ml 0-001 0-01 0-1 1
Control
Treated
18-1 + 4-8 30-7 + 3-4 72-8 + 8-0 100
77-3 + 13-3* 95-7± 4-2** 90-2± 6-7 86-9± 6-9
Lipolysis stimulation by increasing concentrations of synacthen (an analogue of the pituitary peptide ACTH) in adipocytes from control or treated rats after 9 weeks of diet applied since 4 weeks of age. Results are expressed in "to of maximal lipolysis (2- 3 ±0-1 versus 3-0±0-4pmoles glycerol released/100 mg lipid/60 min in control and treated, respectively, mean +SEM, n = 3). Difference between treated and control: * p < 0-05; **/?< 0-001.
SI36
/. Harant et al. Table 2. Basal and insulin-stimulated lipogenesis of adipocytes from control, EFA deficient, and LA supplemented rats. nmoles glucose/100 mg lipid/120 min Feeding condition Control Deficient Supplemented LA 1% Supplemented LA 2-5% Supplemented LA 5%
Basal 31-5+ 1-1 126-7±23-2* 78-8± 12-4* 46-4±7-8 34-2± 7-0
Insulin (2500 n lU/ml) 81-5± 9-0 312-2±56-2* 186-5±42-4* 91-7±13-5 82-8±12-4
Glucose conversion in triglycerides was measured on isolated adipocytes from rats fed with normolipidic diet (control), unsaturated FA-free diet for 9 weeks (deficient), or unsaturated FA-free diet followed by dietary LA supplementation for 4 weeks (supplemented). Results are expressed in nmoles of glucose incorporated into cellular Hpids/lOOmg lipids/120min. Mean ±SEM of three determinations. Differences between treated and control: * p < 0-05.
hypoglycaemic after 5 weeks of treatment. These results (increase of lipogenic activity and hypoglycaemia) are in good agreement with those observed by Berdanier and Baltzell (1986). After 9 weeks of EFA-deficient diet we supplemented the deficient rats by addition of LA to the diet for 6 weeks. The correction of the EFA deficiency symptoms was studied as a function of the level of supplementation in LA: 1,2-5 or 5% of total calorie intake. Supplementation with LA induced a rapid 'normalization' of body weight which returned to control values after 6 weeks except for the group supplemented with only 1%. Whatever the level of LA supplementation, adipose mass reached control values within 4 weeks. So, during the first four weeks of supplementation, there was a 2-fold increase in the adipose tissue mass. Such a propensity of EFAdeficient rats to enlarge the fat depots could be the result of the increased capacity of fat cells to consume glucose in vitro, as reported above. The addition of LA to the diet reversed, in a dose-dependent manner, the alterations of erythrocyte and adipocyte FA composition induced by EFA deficiency. The FA composition of erythrocytes and adipocytes returned to normal values within 4 weeks of LA supplementation at 5% and within 6 weeks for the group at 2-5%. By contrast, for the group at 1%, 6 weeks were not sufficient for a complete restoration of these parameters. Lipolytic activity returned to control values in the 3 groups of supplemented animals. The insulin-hyperresponsiveness disappeared within 4 weeks for groups at 2-5 and 5% (table 2). The diminution was progressive for the group at 1% and returned to the control value after 6 weeks of treatment. Conclusions
In conclusion, the absence of dietary unsaturated FA provokes an important reduction of adipose tissue development which is preceded by alterations of the FA composition of adipocyte membranes and TG stores. These alterations are similar to the modifications of blood FA patterns described in EFA deficiency. They are
Linoleic acid and rat adipocyte metabolic disorders
S137
accompanied by an increase in ACTH sensitivity and a greater basal and insulinstimulated lipogenesis. These observations are totally reversed in 4 weeks by the addition of 5% of LA in the unsaturated FA-deficient diet and in 6 weeks with 2-5% of LA, whereas addition of 1% of LA only reversed partially these phenomena in the same time. References BERDANIER, C. D., and BALTZELL, J. K., 1986, Comparative studies of the responses of two strains of rats to an essential fatty acid deficient diet. Comparative Biochemistry and Physiology, 85(A), 725-727. GARCIA, J.,
GHISOLFI, J.,
LAPALU-TRAON, C.,
PERIQUET, B. OLIVES, J. P.,
BOYER, M.
J.,
and
THOUVENOT, J. P., 1986, Dépistage d'une carence en acides gras essentiels chez l'enfant. Intérêt respectif du dosage des acides gras dans les lipides totaux et les fractions lipidiques du sérum. Annales de Biologie Clinique, 44, 380-383. GOURLEY, G. R., FARRELL, P. M., and ODELL, G. B., 1982, Essential fatty acid deficiency after hepatic portoenterostomy for biliary atresia. American Journal of Clinical Nutrition, 36, 1194-1199. JUANEDA, P., and ROCQUELIN, G., 1985, Rapid and convenient separation of phospholipids and nonphosphorus lipids from rat heart using silica cartridges. Lipids, 20, 40-41. KRITCHEVSKY, D., TEPPER, S. A., LLOYD, L. M., DAVIDSON, L. M., and KLURFELD, D. M., 1988, Serum
and liver lipids of rats fed cocoa butter, corn oil, palm kernel oil, coconut oil and cholesterol. Nutrition Research, 8, 287-294. MOODY, A. J., STAN, M. A., STAN, M., and GLIEMANN, J., 1974, A simple free fat cell biosassay for
insulin. Hormone and Metabolic Research, 6, 12-16. RODBELL, M., 1964, Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. Journal of Biological Chemistry, 239, 375-380. YAMANAKA, W. K., CLEMANS, G. W., and HUTCHINSON, M. L., 1981, Essential fatty acids deficiency in humans. Progress in Lipid Research, 19, 187-215.
Correction by dietary linoleic acid of rat adipocyte metabolic disorders in essential fatty acid deficiency I. HARANT, C. CARPÉNɆ, J. GARCIA, J. P. THOUVENOT and J. GHISOLFI Groupe d'Etudes en Nutrition Infantile, CEDEM, Hôpital Purpan, 31059 Toulouse, France, and † INSERM U 317, Institut de Physiologie, rue F. Magendie, 31400, Toulouse, France
Introduction
Essential fatty acid (EFA) deficiency occurs in some infant nutritional or digestive diseases such as biliary atresia (Gourley et al. 1982). In order to determine the adequate level of linoleic acid (LA) intake necessary to prevent EFA deficiency symptoms, the effects of LA supplementation on fat cell lipid composition and lipolytic/lipogenic activities were studied in rats submitted to an EFA-deficient diet. Moreover, fatty acid (FA) composition of adipose tissue, a key organ for FA storage, was compared to the commonly used plasma index of EFA deficiency: eicosatrienoate/arachidonate ratio in erythrocyte phospholipids (PL). Materials and methods
Four-week-old rats were submitted to a diet containing 5% lipids (% dry weight) constituted of either peanut- and rape-oil (controls, LA = 2-5% of calorie intake) or the saturated FA: stearate and palmitate (treated, LA = 0-02% of calorie intake). After 9 weeks, the treated rats were divided into three groups and their diets supplemented with different amounts of LA: 1, 2-5 and 5% of calorie intake for 6 weeks. The following parameters were measured: Body weight, adipose mass and blood levels of various metabolites. FA composition of erythrocyte PL: the PL fractions were separated by thinlayer chromatography (Garcia et al. 1986). FA composition of stored triglycerides (TG) and membrane PL of isolated adipocytes: neutral lipids were separated from the PL by the method of Juaneda (1985). After transesterification, FA of erythrocytes and adipocytes were analysed by gas-chromatography (Garcia et al. 1986). In vitro metabolic activity of adipocytes: lipolytic activity (Rodbell 1964) and lipogenic activity (incorporation of (3-3H)-glucose into lipid metabolites according to Moody et al. 1974). Results and discussion
The unsaturated FA-deficient diet induced, as quickly as 2 weeks of treatment, a significant reduction in body weight gain of the rats. After 9 weeks of treatment, body weight was 334 ± 7 g for the treated compared with 421 ± 6 g for the controls (n = 30, p < 0-001). This diminution of growth is a symptom of EFA deficiency as 0265-203X/90 $3.00© 1990 Taylor & Francis Ltd.
Linoleic acid and rat adipocyte metabolic disorders
S135
already reported (Yamanaka et al. 1981, Kritchevsky etal. 1988). The adipose tissue weight was also reduced. After 5 weeks of treatment it was reduced by 50% and more severely after 9 weeks (9-3 ± 1-3 g versus 24-0 ± 2-2 g, p < 0-001). The mass of other organs (brain, liver, heart, pancreas, kidney) were not so affected by EFA deficiency (Berdanier and Baltzell 1986, Kritchevsky et al. 1988, Yamanaka et al. 1981). So, it appears that the EFA deficiency had a specific inhibitory effect on the adipose tissue development. FA composition of erythrocyte PL showed an important decrease of linoleic and arachidonic acid percentages in treated rats. Conversely, the eicosatrienoic acid percentage increased. So, the eicosatrienoate/arachidonate ratio was higher after nine weeks in treated than in control (1-5 ±0-2% versus 0-010 ± 0-002%, p < 0-001). These modifications of erythrocyte FA composition clearly indicated the onset of an EFA deficiency (Yamanaka et al. 1981). In adipocytes, FA composition of stored TG and membranes PL followed the same evolution pattern (C20:3w9/C20:4co6 = 1 -8 ± 0-3% versus 0-08 ± 0-01% for PL in treated and controls, p < 0-001, n = 4). In order to determine if the fat stores reduction was due to an increased lipolytic activity or to a decreased lipogenesis accompanying the modifications of adipocyte membrane composition, these activities were studied in isolated fat cells. No significant change was found in the responses to various lipolytic agents (isoproterenol, forskolin) but there was an increase in sensitivity to ACTH for deficient rats at 5 and 9 weeks of treatment (table 1). This increase of lipolysis, observed in vitro, is in good agreement with the depletion of adipose tissue observed in vivo. However, it is insufficient to explain the adipose mass reduction of treated rats. By contrast, basal and insulin-stimulated lipogenesis were significantly increased in deficient rats (table 2). This observation in vitro is apparently in contradiction with the in vivo fat store depletion observed in the treated group since fat cells with an increased lipogenic activity would correspond to an enlargement of adipose tissue. So, the endocrine or metabolic environment of adipose tissue might be involved in this slimming effect of EFA deficiency rather than a decline in metabolic rate of fat cells. On this point, it must be noted that deficient rats were
Table 1. Effect of unsaturated fatty acid deficiency on lipolytic response of rat adipocytes to synacthen. Percentage of maximal lipolysis Addition to medium synacthen /ig/ml 0-001 0-01 0-1 1
Control
Treated
18-1 + 4-8 30-7 + 3-4 72-8 + 8-0 100
77-3 + 13-3* 95-7± 4-2** 90-2± 6-7 86-9± 6-9
Lipolysis stimulation by increasing concentrations of synacthen (an analogue of the pituitary peptide ACTH) in adipocytes from control or treated rats after 9 weeks of diet applied since 4 weeks of age. Results are expressed in "to of maximal lipolysis (2- 3 ±0-1 versus 3-0±0-4pmoles glycerol released/100 mg lipid/60 min in control and treated, respectively, mean +SEM, n = 3). Difference between treated and control: * p < 0-05; **/?< 0-001.
SI36
/. Harant et al. Table 2. Basal and insulin-stimulated lipogenesis of adipocytes from control, EFA deficient, and LA supplemented rats. nmoles glucose/100 mg lipid/120 min Feeding condition Control Deficient Supplemented LA 1% Supplemented LA 2-5% Supplemented LA 5%
Basal 31-5+ 1-1 126-7±23-2* 78-8± 12-4* 46-4±7-8 34-2± 7-0
Insulin (2500 n lU/ml) 81-5± 9-0 312-2±56-2* 186-5±42-4* 91-7±13-5 82-8±12-4
Glucose conversion in triglycerides was measured on isolated adipocytes from rats fed with normolipidic diet (control), unsaturated FA-free diet for 9 weeks (deficient), or unsaturated FA-free diet followed by dietary LA supplementation for 4 weeks (supplemented). Results are expressed in nmoles of glucose incorporated into cellular Hpids/lOOmg lipids/120min. Mean ±SEM of three determinations. Differences between treated and control: * p < 0-05.
hypoglycaemic after 5 weeks of treatment. These results (increase of lipogenic activity and hypoglycaemia) are in good agreement with those observed by Berdanier and Baltzell (1986). After 9 weeks of EFA-deficient diet we supplemented the deficient rats by addition of LA to the diet for 6 weeks. The correction of the EFA deficiency symptoms was studied as a function of the level of supplementation in LA: 1,2-5 or 5% of total calorie intake. Supplementation with LA induced a rapid 'normalization' of body weight which returned to control values after 6 weeks except for the group supplemented with only 1%. Whatever the level of LA supplementation, adipose mass reached control values within 4 weeks. So, during the first four weeks of supplementation, there was a 2-fold increase in the adipose tissue mass. Such a propensity of EFAdeficient rats to enlarge the fat depots could be the result of the increased capacity of fat cells to consume glucose in vitro, as reported above. The addition of LA to the diet reversed, in a dose-dependent manner, the alterations of erythrocyte and adipocyte FA composition induced by EFA deficiency. The FA composition of erythrocytes and adipocytes returned to normal values within 4 weeks of LA supplementation at 5% and within 6 weeks for the group at 2-5%. By contrast, for the group at 1%, 6 weeks were not sufficient for a complete restoration of these parameters. Lipolytic activity returned to control values in the 3 groups of supplemented animals. The insulin-hyperresponsiveness disappeared within 4 weeks for groups at 2-5 and 5% (table 2). The diminution was progressive for the group at 1% and returned to the control value after 6 weeks of treatment. Conclusions
In conclusion, the absence of dietary unsaturated FA provokes an important reduction of adipose tissue development which is preceded by alterations of the FA composition of adipocyte membranes and TG stores. These alterations are similar to the modifications of blood FA patterns described in EFA deficiency. They are
Linoleic acid and rat adipocyte metabolic disorders
S137
accompanied by an increase in ACTH sensitivity and a greater basal and insulinstimulated lipogenesis. These observations are totally reversed in 4 weeks by the addition of 5% of LA in the unsaturated FA-deficient diet and in 6 weeks with 2-5% of LA, whereas addition of 1% of LA only reversed partially these phenomena in the same time. References BERDANIER, C. D., and BALTZELL, J. K., 1986, Comparative studies of the responses of two strains of rats to an essential fatty acid deficient diet. Comparative Biochemistry and Physiology, 85(A), 725-727. GARCIA, J.,
GHISOLFI, J.,
LAPALU-TRAON, C.,
PERIQUET, B. OLIVES, J. P.,
BOYER, M.
J.,
and
THOUVENOT, J. P., 1986, Dépistage d'une carence en acides gras essentiels chez l'enfant. Intérêt respectif du dosage des acides gras dans les lipides totaux et les fractions lipidiques du sérum. Annales de Biologie Clinique, 44, 380-383. GOURLEY, G. R., FARRELL, P. M., and ODELL, G. B., 1982, Essential fatty acid deficiency after hepatic portoenterostomy for biliary atresia. American Journal of Clinical Nutrition, 36, 1194-1199. JUANEDA, P., and ROCQUELIN, G., 1985, Rapid and convenient separation of phospholipids and nonphosphorus lipids from rat heart using silica cartridges. Lipids, 20, 40-41. KRITCHEVSKY, D., TEPPER, S. A., LLOYD, L. M., DAVIDSON, L. M., and KLURFELD, D. M., 1988, Serum
and liver lipids of rats fed cocoa butter, corn oil, palm kernel oil, coconut oil and cholesterol. Nutrition Research, 8, 287-294. MOODY, A. J., STAN, M. A., STAN, M., and GLIEMANN, J., 1974, A simple free fat cell biosassay for
insulin. Hormone and Metabolic Research, 6, 12-16. RODBELL, M., 1964, Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. Journal of Biological Chemistry, 239, 375-380. YAMANAKA, W. K., CLEMANS, G. W., and HUTCHINSON, M. L., 1981, Essential fatty acids deficiency in humans. Progress in Lipid Research, 19, 187-215.
